Method for purifying GBS toxin/CM101

ABSTRACT

A method for purifying a polysaccharide from group B β-hemolytic Streptococcus (GBS) bacteria includes contacting a bacterial fermentation stock with a hydrophobic interaction chromatography (HIC) resin. Additional steps may include a phenol/saline extraction and an ion exchange chromatography. The method results in a product having very high purity. The product of the purification provides a composition which is highly useful in both research and therapeutic settings.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national stage application of InternationalApplication No. PCT/US97/17535, filed Sep. 30, 1997, which is acontinuation of U.S. Ser. No. 08/744,770, filed Sep. 30, 1996 and issuedas U.S. Pat. No. 5,811,403 on Sep. 22, 1998.

TECHNICAL FIELD

This invention relates to improved methods of purification for apolysaccharide.

BACKGROUND

CM101, a GBS toxin, is a pathogenic molecule isolated from group Bβ-hemolytic Streptococcus (GBS) bacteria. Newborn infants may becomeinfected with GBS, a condition known as GBS pneumonia or “early-onsetdisease,” and suffer from sepsis, granulocytopenia, and respiratorydistress, i.e. pulmonary hypertension and proteinaceous pulmonary edema(Hellerqvist, C. G. et al., Studies on group B β-hemolytic streptococcusI. Isolation and partial characterization of an extra-cellular toxin.,Pediatr. Res., 15:892-898 (1981)).

Despite the harmful effects to neonates exposed to GBS, CM101 is notknown to cause toxicity in older humans. In fact, research into thistoxin has revealed a significant therapeutic application. See U.S. Pat.No. 5,010,062 and Hellerqvist, C. G. et al., Early Results of a Phase ITrial of CM101 in Cancer Patients., Proceedings of the AmericanAssociation of Cancer Research Annual Meeting (1995), wherein CM101 isutilized to inhibit vascularization of tumors. Obtaining purified CM101is critical, therefore, for both research and therapeutic purposes.

CM101 is a complex polysaccharide toxin having a molecular weight ofapproximately 300,000 Daltons and comprising N-acetyl-galactosamine,N-acetyl-glucosamine, glucose, galactose, and mannose residues. Nmr(nuclear magnetic resonance) results suggest that alditol residues mayalso be present. Carboxylic acid functional groups, probablygalacturonic acid, are also believed to be an integral part of themolecule. Repeating active epitopes most likely play an important rolein the pathophysiological response to CM101 by crosslinking receptors ontarget endothelium (Hellerqvist, C. G. et al. Early Results of a Phase ITrial of CM101 in Cancer Patients., Proceedings of the AmericanAssociation of Cancer Research Annual Meeting (1995); DeVore, R. F., etal., A Phase I Study of the Antineovascularization Drug CM100, J. Clin.Can. Res., 3:365-372 (1997)).

U.S. Pat. No. 5,010,062 provides a method of purification of a GBStoxin. The method taught is labor-intensive. however, requiring numeroussteps with continual levels of loss of biological activity.

Purification of CM100 as presently known in the art provides an endmaterial which is only 40% pure as measured by chemical analyses andbiological assays. The other 60% comprises plant and yeastpolysaccharides and endogenous bacterial polysaccharides. The plant andyeast contaminants originate for the most part in the additives to thecommercial culture media used for optimal growth of the GBS bacteria.The endogenous contaminants include GBS polysaccharides including groupand type specific antigens (Paoletti, L. C. et al., Neonatal mouseprotection against infection with multiple group B streptococcal (GBS)serotypes by maternal immunization with a tetravalent GBSpolysaccharide-tetanus toxoid conjugate vaccine, Infect. Immun.62(8):3236-43 (1994); Michon, F., Multiantennary group-specificpolysaccharide of Group B Streptococcus, Biochem., 27:5341-51 (1988)).CM100 of this 40% purity level represents the current clinical grade.There is a need, therefore, for a purification method of CM100 whichresults in an end product with increased overall purity, preferably withthe removal of extraneous plant and yeast polysaccharides and GBSantigenic polysaccharides.

Additionally, the purification scheme known in the art includesenvironmentally unsound steps, such as the use of a large volume ofphenol in a phenol:water extraction. Phenol is a well-known causticmaterial.

Therefore, objects of the present invention are to provide apurification method resulting in (i) a material of high purity, (ii)using a minimal number of steps, (iii) minimizing the use of caustic ortoxic materials such as phenol, and (iv) increasing the yield ofmaterial.

SUMMARY OF THE INVENTION

The above objects have been achieved with the invention describedherein. Particularly, a purification scheme including a hydrophobicinteraction chromatography (HIC) resin for purification of CM101 fromGBS bacterial culture media results in a product of greater than 95%purity.

One aspect of this invention is a process for purifying a polysaccharidetoxin from GBS bacteria, the process including the use of an HIC resin.The present invention also includes a substantially pure polysaccharidetoxin from GBS bacteria produced by the method disclosed herein, and apharmaceutical composition comprising a substantially pure toxin and apharmaceutically acceptable carrier. The pharmaceutical composition maybe used to treat a patient having a medical condition. For example, atumor patient may be treated with the composition of the presentinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a CM101 purification scheme of the present invention.

FIG. 2 illustrates a known CM101 purification scheme.

FIGS. 3a-3 c are quantitative hydrolysis standard curves showing thedose response of a PAD detector for 5 μg (FIG. 3a), 20 μg (FIG. 3b), and50 μg (FIG. 3c) of dextran (a glucose polymer) with 6-deoxy glucose as aconstant internal standard.

FIG. 4 shows the separation of standard sugar samples.

FIGS. 5a-b are elution profiles of a media concentrate on abutyl-Sepharose HIC column. FIG. 5a is measured at UV 206 absorbance.FIG. 5b is measured at UV 280 absorbance.

FIG. 6a is an HPLC-profile of an HIC-purified water-eluted fractioncontaining CM101 (16 min peak) and monitored at UV 203 absorbance on aMillennium 2000 Diodo-Ray detector (Waters, Millford, Mass.).

FIG. 6b is a Diodo-Ray spectrum corresponding to FIG. 6a andillustrating minimal presence of 260 absorption (RNA and DNA) and 280absorption (tyrosine-containing protein) for the CM100 containing (16min) peak.

FIG. 7a is an elution profile monitored at 203 nm showing the purity ofthe HIC water-eluted peak of FIG. 6a further subjected to phenol/salineextraction and subsequent DEAE chromatography.

FIG. 7b is a Diodo-Ray spectrum illustrating the purity of theCM101-containing peak of FIG. 7a as evidenced by the narrow symmetricpeak and the lack of absorption at 260 nm (RNA/DNA) and 280 nm(protein).

FIG. 8 is a profile of IL-6 activity by ANA-1 Assay of fractionsobtained from an HIC column, more specifically an IL-6 activity profileof fractions obtained from 10K5P6 concentrate run on 100 ml ButylSepharose (FT=flow-through; 1M=1M phosphate fraction; 0.25M 0.25Mphosphate fraction: H₂O=water fraction: EtOH=ethanol fraction).

FIG. 9 illustrates a sugar analysis of CM101 purified by the method ofthe present invention.

FIG. 10 is an HPLC profile of current clinical grade CM101 furthersubjected to HIC chromatography.

FIG. 11 illustrates a sugar analysis of a sample of current clinicalgrade CM100 which was further purified by HIC and HPLC.

FIG. 12a is an HPLC profile of CM100 purified by a known process using10 mM phosphate buffer. pH 8.4.

FIG. 12b is an HPLC profile of CM100 purified by the method of theinvention, using the same running conditions as the HPLC profile of FIG.12a.

DESCRIPTION OF SPECIFIC EMBODIMENTS

GBS toxin as used herein is defined as any fraction or componentisolated from natural or lysed GBS bacteria, or derived from mediasupernatants of lysed and/or autoclaved GBS bacteria. and which has abiological activity evidenced by induction of respiratory distress inthe sheep assay (Hellerqvist, C. G. et al., Studies on group Bβ-hemolytic streptococcus I. Isolation and partial characterization ofan extra-cellular toxin., Pediatr. Res., 12:892-898 (1981)) oractivation of complement and binding to neovasculature as demonstratedby a peroxidase-antiperoxidase (PAP) assay of a tumor tissue specimen(Hellerqvist, C. G. et al., Anti-tumor effects of GBS toxin: apolysaccharide exotoxin from group B β-hemolytic streptococcus, J. CancRes. Clin. Oncol., 120:63-70 (1993); and Hellerqvist, C. G. et al.,Early Results of a Phase I Trial of CM100 in Cancer Patients.,Proceedings of the American Association of Cancer Research AnnualMeeting (1995)).

Substantially pure GBS toxin means a preparation in which GBS toxin isgreater than 40% pure (e.g., present in a concentration of at leastabout 40% by weight), preferably at least approximately 60% pure, morepreferably at least approximately 90% pure. and most preferably at leastapproximately 95% pure.

A source for GBS starting material for use in the method of the presentinvention may be obtained by culturing strains of Group B β-hemolyticStreptococcus bacteria that have recently infected or are capable ofinfecting newborn infants. Isolates of such strains may be obtained fromthe blood of infected infants.

High production of CM101 generally requires fermentation with thecomplex media THB which contains high molecular weight material in theform of polysaccharides and proteins for GBS optimum growth and CM101production. During the fermentation process. the bacteria produce fromthe nutrients quantities of proteins, nucleic acids, and polysaccharidesother than CM100. The estimated concentration of CM101 in thefermentation broth is less than 0.1% by weight.

The purification method of the present invention employs hydrophobicinteraction chromatography (HIC) which eliminates the bulk of theendogenous and exogenous contaminating proteins. nucleic acids andpolysaccharides more efficiently than known methods and results in anend product which contains 10-50% pure CM101. In just one step ofcontacting the GBS starting material and the HIC resin, this representsa 100-500 fold purification from the starting material.

Use of an HIC resin for purification of a polysaccharide is surprisingand novel because HIC columns arc designed for purification ofhydrophobic proteins and are not believed useful for polysaccharidesfree of proteins and lipids. Polysaccharides are generally characterizedas being hydrophilic due to their numerous hydroxyl groups. Applicationof a starting material to an HIC column under the conditions recommendedby the manufacturer and used by practitioners skilled in the art wouldtherefore be with the intention of retaining proteins and allowingpolysaccharides to pass through the column unbound.

The surprising discovery is that CM100 has hydrophobic properties thatallow use of the present purification scheme to achieve a high level ofpurity. Especially surprising is that CM100 has significantly morehydrophobic characteristics than most of the proteins andpolysaccharides present in the supernatant from which the CM100 isisolated. Greater than 98% of these protein and polysaccharidecontaminants pass through the HIC column. Although the HIC resin isgenerally employed in an HIC column, this step alternatively may beperformed by contacting the resin and the starting material in someother manner. For example. the GBS source and the resin may be placed ina vessel together in a batchwise process, and the toxin-containingportion subsequently separated from the resin as by centrifugation.

Additional purification steps may include a phenol/saline extraction ina small volume relative to the prior methods (approximately 1000-foldreduced) and an ion exchange column. These additional purification stepscontribute to an end product with greater than 95% purity.

HIC is a method used to separate proteins, such as membrane proteins,based on their hydrophobic nature. An HIC resin is defined as a resinhaving interactive hydrophobic groups which are generally covalentlyattached to a support such that the hydrophobic groups are free tointeract with substances in contact with the resin. Examples ofhydrophobic groups include alkyl, alkoxy, and aryl groups. The preferredHIC resin to be used in accordance with the present invention has asupport with attached aliphatic groups of two or more carbons,preferably alkyl groups in the range of 2 to 12 carbons, and morepreferably normal or branched butyl groups. Phenyl groups or alkoxygroups of up to 20 carbons are also preferred interactive hydrophobicgroups. The interactive hydrophobic groups are preferably supported bySepharose (Pharmacia), acrylamide (Toso Haas, Montgomeryville, Pa.), orsilica. According to the standard procedure for use of an HIC column,the starting material containing the protein of interest is applied tothe column in up to 2M aqueous salt solution and the bound proteins arethen eluted and separated through decreases in hydrophobic interactionsby reducing the ionic strength of the developing buffer. Changes in pHand/or temperature may also be used to alter the hydrophobicinteractions.

CM101 purification from Group B Streptococcus requires obtaining abacterial culture of GBS. Bacterial inocula are incubated to late logphase in Todd Hewitt Broth (THB) modified by supplementation with 2 g/lor more of glucose and Na₂HPO₄. As indicated in FIG. 1, the culture isthen autoclaved. CM101 is present in the supernatant of GBS fermentationcultures at a concentration of 2-15 mg/l following autoclaving. Themedia contains approximately 15 g/l of other bacterial and mediacomponents. Thus, CM101 constitutes approximately 0.01-0.1% of thecomponents in the supernatant. After autoclaving, the media is filtered.The filtrate is preferably concentrated via a 10,000 Dalton (10 kD)cutoff filter, although a filter having a cutoff of 50,000 Dalton (50kD) or less may be used.

CM101 is then purified in accordance with this invention. as shown inFIG. 1, by applying a supernatant concentrate or reconstituted alcoholprecipitate thereof made 0.75-2M in potassium phosphate or another salt,such as sodium phosphate, sodium or potassium sulphate. chloride, oracetate, to an HIC column preferably equilibrated in the same salt ofthe same molarity. The method wherein the media concentrate, or 10 kD-50kD, starting material is employed without alcohol precipitation andreconstitution is preferred because the media concentrate startingmaterial provides higher yield of CM100 than does the reconstitutedalcohol precipitate.

The CM101-containing starting material is applied to the HIC column andwashed with aqueous phosphate at 0.75-2M. Following a 0.75-2M wash, thecolumn is further developed with 0.5-1M and then 0.25M salt, preferablyphosphate. In the preferred embodiment. the CM100 is eluted from thecolumn with water as a single peak containing 10-50% CM101.Alternatively, water is replaced for CM100 elution from the HIC columnwith 10 mM phosphate. pH 6.8 in 10% ethanol in water (Buffer A),followed by 20% ethanol in water. CM101 activity is recovered in boththe Buffer A and 20% ethanol fractions. Use of Buffer A is generally notsufficient to remove all the CM100 from the HIC column, so the Buffer Awash is followed by an additional 20% ethanol wash. However, inscale-up, the ethanol constitutes an environmental hazard and thesubsequent phenol/saline extraction of the water peak or the Buffer Aand 20% ethanol peak fractions yields CM100 of approximately equalpurity. The HIC procedure removes better than 98% of both the proteinsand media polysaccharides remaining in the 10 k concentrate or thereconstituted alcohol precipitate.

The enriched CM100 from the HIC column may be further purified by anextraction in phenol and an aqueous salt solution, preferably 0.05Msaline. This additional step provides a CM100 fraction of approximately95% purity.

The water or the combined Buffer A and 20% ethanol fractions eluted fromthe HIC column are either dialyzed against water and lyophilized andreconstituted in 0.05M saline or dialyzed against saline afterconcentration. Typically, phenol is added to the material and thesolution is rapidly heated to 70-80° C. When a single phase forms. thesolution is chilled to 4° C. The resulting saline phase of thephenol/saline extraction contains CM101 and may then be applied to acation exchange column, such as DEAE.

For the DEAE column procedure, the DEAE column is equilibrated in waterand then washed with 0.1M saline, 0.05M NaOAc, pH 7.4 and developed witha step gradient to 0.34M NaCl. Elution of CM101 is monitored andquantitated with an ANA-1 assay. As with the HIC resin step, an ionexchange resin may be contacted with the toxin-containing materialthrough use of equipment other than a column. The CM101-containingfraction is then dialyzed against water and lyophilized. After thephenol/saline extraction and ion exchange steps, CM101 is greater than95% pure.

The column eluates, or material resulting from the resin contact steps,are assayed for biologic activity with the ANA-1 and/or Dot Blot assay.The biological activity is then confirmed with a sheep assay. Table 1depicts several separations of AP and 10 k material obtained fromdifferent batches of starting materials and applied to the HIC column.Removal of denatured protein and media polysaccharides and othermaterial is similar.

Although the preferred order of purification is to perform the HIC step,followed by the phenol/saline extraction, and then the ion exchangestep, purification may also be performed in another order.

FIG. 2 presents an example of a known method of CM100 purification.Notably, a 70% ethanol precipitate step is used. followed soonthereafter with a phenol/water extraction. The large volumes of ethanoland phenol required at these early stages of the known purificationmethod represent environmentally unsound practices. The methodrepresented in FIG. 2 also requires an ion exchange column, a gelfiltration column, and a lentil lectin column.

The prior method contains numerous steps. including environmentallyhazardous ones. On the other hand, the method of the present inventionis effective, gives higher purity and 2 to 25 times the yield, andminimizes use of environmentally unsound materials.

The environmentally hazardous phenol-water extraction step is reduced1000-fold as compared to the previously used procedures. Furthermore,additional purification as by a gel filtration procedure is eliminated.The lentil lectin chromatography step of the prior method is alsodeleted. The end product of the HIC column , phenol/saline extraction,and ion exchange column steps has approximately 95% purity, so othertreatments are unnecessary.

The CM100 purified by the method of the present invention may be usedfor research or therapeutic purposes. The CM101 is particularly usefulwhen combined with a pharmaceutically acceptable carrier, e.g.,reconstituted in saline and administered to a patient intravenously.Other dosage forms to administer purified CM101 may also be used. Thepharmaceutical composition of this invention comprises the substantiallypure GBS toxin of this invention in in combination with apharmaceutically acceptable carrier. In general. the carrier will be onethat is readily mixed with the toxin to form a composition that isadministrable by intravenous (IV) means. Thus, the carrier is preferablywater. which may have other pharmaceutically acceptable excipientsincluded to ensure its suitability for intravenous administration. Theresulting composition will be sterile and will have acceptable osmoticproperties In general, a suitable IV formulation is prepared inaccordance with standard techniques known to one of skill in the art.For example, Chapter 85 entitled “Intravenous Admixtures” by SalvatoreJ. Turco in the Eighteenth Edition of Remington's PharmaceuticalSciences, Mach Publishing Co. (1990), incorporated herein by reference,provides standard techniques for preparing a pharmaceutically acceptableIV composition useful in accordance with this invention.

Additionally, a patient having a medical condition which is found torespond advantageously to CM101 may be treated with a pharmaceuticalcomposition of the present invention. For example, a patient having atumor may be advantageously treated by intravenously administering thepharmaceutical composition taught herein. U.S. Pat. No. 5,010,062discusses the treatment of certain tumors in humans and is incorporatedherein by reference.

Quantitative and Qualitative Analysis HPLC Analysis

The purity and amount of CM101 obtained from a sample after HICchromatography is established by high pressure liquid chromatograph(HPLC) gel filtration analysis. The gel filtration column is typicallyequilibrated with 10% acetonitrile in water and the biologically activeCM101 is eluted as an included homogeneous narrow peak. Alternatively,the column may be developed in 10 mM phosphate buffer, pH 8.4, whichyields a more included peak. An ammonium acetate (NH₄OAc) buffer, pH8.4. may be used as a further alternative to the 10 mM phosphate buffer.

A typical detector response (UV 203 absorption) using 30. 50, and 10 μgpure CM101 standards injected in 100 μl of developing buffer on aHydragel 1000 column (Waters, Millford. Mass.) is 26×10⁶, 48×10⁶, and97×10⁶ area units, respectively which yields a do se e curve forquantitation of unknown samples.

The molecular weight of CM100 may also be measured by gel filtrationchromatography. A non-denaturing buffer such as the acetonitrile.phosphate or ammonium acetate buffers described above are used to runthe column. The CM101 elution is compared to that of standard dextranpolysaccharide markers of different molecular weight. The CM100 has amolecular weight of approximately 300,000 Daltons under theseconditions.

Amino Acid Analysis

Quantitative and qualitative automated amino acid analysis may beperformed with standard commercially available equipment. e.g. PicoTag.available from Waters. Millford. Mass.

ANA-1 Assay

To monitor the biological activity of the different fermentation andpurification steps, an in vitro assay employing a transformed mousemacrophage cell line may be used. The assay measures IL-6 production ofthe mouse macrophage ANA-1 in response to CM100 exposure.

Particularly, CM100 induces raf/myc transformed murine bone marrowmacrophage cell line ANA-1 to respond in vitro by IL-6 production. Othermacrophage-like cell lines and fresh peripheral blood leukocytes canalso be used.

To perform the ANA-1 assay, samples are first diluted to the appropriaterange (depending on the expected level of CM101 activity) and four toeight concentrations are tested at 1:4 dilutions. A CM100 standard curveusing clinical grade CM100 reconstituted in PBS is generated. A 4000ng/ml solution, which gave a 2000 ng/ml final concentration after thecells were added, was made in PBS. along with six serial 1:2 dilutions.Cells at a concentration of 2×10⁶/ml may be used, for example.Sensitivity of the assay was increased by adding 200 U/ml murine IFN-γto the ANA-1 cells. Final cultures were 100 U/ml IFN-γ.

The microtiter plate with cultures should be placed in a 37°, 5%CO₂-in-air, humidified incubator overnight (16-18 hours), and then befollowed by an ELISA IL-6 Assay (R.D. Systems. Minneapolis, Minn.).Specifically, culture supernatants are transferred to the IL-6 assayplate and the plate is held at 4° C. until the IL-6 assay is complete.

Dot Blot Assay

An alternative rapid procedure to quantitatively detect CM100 insolutions or biological fluids is to blot samples on polyvinylidinedifluoride (PVDF) membranes in serial dilutions. The amount of CM100 isquantitated using either a fluorescently-tagged mouse monoclonalantibody to CM100 or a mouse monoclonal antibody to CM101 followed by afluorescently-tagged anti-mouse IgG. Antibody 7A3 directed against CM100antigen is useful for this purpose. Quantities of CM100 in the differentfractions are established by comparison to a standard curve of serialdiluted CM100 standard.

Sheep Pulmonary Arterial Pressure Assay

The toxin affects sheep lungs by increasing pulmonary hypertension.manifested by increased pulmonary arterial pressure and by increasedlung vascular permeability.

CM101 samples in phosphate buffered saline (PBS) may be administered tolambs by infusion and changes in pulmonary arterial pressure recorded at15 minute intervals. These changes in pressure are correlated to CM101activity. (Hellerqvist. C. G. et al., Studies on group B β-hemolyticstreptococcus I. Isolation and partial characterization of anextra-cellular toxin., Pediatr. Res., 15:892-898 (1981)).

Sugar Analysis

A 100 μg quantity of a sample is hydrolyzed for two hours at 100° C. ina mixture of trifluoroacetic acid (TFA), acetic acid (HOAc) and water ina ratio of 5:70:25. The solution is evaporated and the sample is furtherhydrolyzed for two hours at 100° C. in a mixture of TFA and water in aratio of 2:8. This process completely hydrolyses all glycosidic linkagesin the sample. The N-acetyl groups originally present on the aminosugars are also removed.

The samples are then analyzed on the Dionex sugar analysis system usinga PAD (Pulsed Amperometric Detection) detector. The resolution isillustrated in FIG. 4.

The purity of the sample is established by quantitative and qualitativesugar analysis. The principle is illustrated in FIGS. 3a- 3 c and FIG.4. A sample of polysaccharide quantitated by HPLC is supplemented withan internal standard 6-deoxy-D-glucose hydrolyzed and analyzed. Themethod described in this section gives a linear dose response in therange tested and qualitative analysis is accomplished by comparingretention times of unknowns with the standards.

EXAMPLES Example 1 A Scaled-up Purification Scheme for CM101

A Group B Streptococcus Serotype III isolate working stock was used inconjunction with a 3,000 gallon fermentor. A 25 ml seed of the bacterialculture is used for an 80 liter vessel with a 65 liter working volume(lwv) which is then used to inoculate a 750 lwv vessel, and which, inturn, goes into the final 7500 lwv (3,000 gallon) fermentor.Alternatively, the 65 lwv may be used to inoculate the 7500 lwvfermentor directly.

The cultures are terminated at late log phase by autoclaving. Thebacteria are then removed by continuous centrifugation at 10,000×g,followed by 0.45 micron cassette filtration (Millipore Corporation,Bedford, Mass.).

The resulting culture supernatant is then concentrated 15-fold throughcassette filtration using 10 kD to 50 kD cut off cassettes (Millipore)to 500 liters. The concentrated material is then made 2M in salt,preferably sodium phosphate, pH 7.4 (loading buffer) by dialysis.

The concentrated supernatant is then subjected to hydrophobicinteraction chromatography, through the use of a 60 liter n-butylSepharose column (Pharmacia, Uppsaia, Sweden) using a BioPilot system(Pharmacia). The capacity of the n-butyl Sepharose resin for thebiologic CM101 activity in the media concentrate with no flow-through ofactivity is approximately 80 liter of media to one liter of resin. Afterthe concentrated supernatant is loaded onto the column, the column iswashed with the loading buffer followed by 0.5-1M and then 0.25Mphosphate buffer. pH 7.4. The CM101-containing fraction is eluted withwater in approximately 120 liters or two column volumes and concentratedto 2 liters in a cut-off cassette in the range of 10 kD to 50 kD. Thecolumn elution is controlled by a preestablished program in the BioPilotand the eluate is monitored by UV absorption at 206 and 280 nm.conductivity, and pH.

The CM101-containing 2 liter fraction is dialyzed against 0.05M saline,pH 7.0 and then heated to the range of 75-80° C. and 0.2-2 liters ofphenol are added. The mixture is then heated to 80° C. and maintained atthat temperature for 5 minutes. Following this, the mixture is chilledto 4° C. The water phase resulting from this step is preferablyextracted twice with 0.2 volumes chloroform before application to a DEAESephacel FF column (Pharmacia, Uppsala, Sweden) equilibrated in water.The column is washed with 100 mM saline, 0.05M NaOAc, pH 7.4, and thebiologically active material CM101, is then eluted from the DEAE columnwith a NaCl gradient. The biological activity is detected by Il-6 assayand HPLC analysis. The quality of the CM101 purified through thisprocedure is established by HPLC and sugar analysis as well asbiological activity assays by Il-6 and sheep tests.

This scaled up purification scheme provides the advantage of avoidingthe large volume, early phenol-water extraction procedure of the alcoholprecipitate used in the previous procedure.

Results

FIGS. 5a-b show elution profiles of a media concentrate on abutyl-Sepharose HIC column in 2M K₂HPO₄, pH 7.2. The various peaks arethe results of timed step-wise changes in the elution gradient. FIG. 5arepresents the profile measured at UV 206 absorbance, which quantitatesthe peak fractions for total organic material, and shows the CM101 inthe last narrow peak (approximately 383 minutes). FIG. 5b represents theprofile measured at UV 280 absorbance, which quantitates the amount ofprotein in the different fractions.

By performing the HIC column step, CM100 is caused to bind to the columnwhereas up to 99.7% of the protein and tip to 98.5% of neutral andcharged polysaccharides pass through the column. as indicated in Table1.

TABLE 1 Purification of CM101 Activity by HIC ChromatographyQuantitation by Integration of UV 280 and 206 Profiles Final ElutionTotal Organic Possible Protein UV280 UV206 Recovered % Recovered % AP6P6 Water 0.85 2.67 AP 2P9 Water 1.08 0.19 10K5P6 Water 0.82 1.05 10K5P6Water 0.46 2.43 AP 1 P9 Buffer A 0.39 1.90 10K5P6 Buffer A 0.50 1.51 AP6P6 Buffer A 0.19 1.35

In Table 1, different fermentation lots as alcohol precipitates (AP),AP1, AP2, and AP6, and 10 k concentrates were subjected to HICchromatography and eluted with either water or Buffer A. Both processesyield approximately the same efficacious removal of exogenous andendogenous protein (UV 280) and polysaccharides and general organics (UV206).

FIGS. 6a-b present an HPLC profile, and a Diodo-Ray spectrum, of anHIC-purified water-eluted fraction containing CM100 and monitored at UV203 absorbance. These figures illustrate the minimal presence of 260absorption (RNA and DNA) and 280 absorption (protein) for the CM100containing peak.

After the HIC fraction is further subjected to the phenol/salineextraction and ion exchange steps, the purity of the HIC water-elutedpeak is further improved, as seen in FIGS. 7a-b. Note the narrowsymmetric peak at approximately 16 minutes from time zero and the lackof absorption at 260 (RNA/DNA) and 280 (protein). For the elutionprofiles shown in FIGS. 6a-b and 7 a-b, the HPLC was performed with 10%acetonitrile in water and the flow rate was approximately 0.3 ml/min.

These elution profiles as well as the biological activity are similar tothose obtained when the alcohol precipitate is used as the startingmaterial for the HIC column.

The ability of the HIC fractions from the 10 k starting material toinduce IL-6 synthesis in ANA-1 cells is illustrated in FIG. 8. HICchromatography yielded an approximate recovery of 50% of the totalbiologic activity in the media supernatant as measured by an ANA-1Assay. Dot blot assays of the same material which show immunoreactivityin the presence of CM100 antigen were used to confirm ANA-1 assayresults.

The different fractions obtained from the 10 k concentrate after HICchromatography were also tested in the sheep model for biologicactivity. The amount of CM101 activity is determined based on a doseresponse curve using current clinical CM101 (1 Unit of activitycorresponds to 7.5 μg/kg). The results are shown in Table 2 wherein HICfractionations of alcohol precipitate (AP) and media concentrate (10 k)are compared.

TABLE 2 Amount of CM101 Obtained from HIC Chromatography of AP and 10KMaterial Based on Quantitation of Biological Activity in Sheep Model(10k) Alcohol Precipitate (AP) Media Concentrate Fraction CM 101Activity μg/l CM 101 Activity μg/l Pre-Load 466 Not Available 1MPhosphate 118  209 0.25 Phosphate 28 2970 Water 225 7520

The biological activity of CM100 as purified by the method of thepresent invention was also measured with the pulmonary arterial pressureassay in sheep, and then compared with the activity of CM100 purified bythe old process. for example as taught in U.S. Pat. No. 5.010,062. Thematerial purified according to the invention exhibited a specificactivity of two to three times greater than material which was purifiedby the old process. that is, which had not been contacted with an HICresin.

The product yield of the method of the present invention is alsoevidenced above, as the known methods provide about 300 μg of CM100 perliter of fermentation volume, as compared with the 7520 μg/l value shownabove.

The purified CM101 illustrated in FIGS. 7a-b obtained by the process ofthe present invention was also subjected to sugar analysis. The sugaryields are shown in FIG. 9.

Quantitatively, the CM101 obtained by the method of the presentinvention is greater than 95% pure carbohydrate and contains less than5% of protein established by quantitative and qualitative as presentedabove and by automated amino acid analysis (PicoTag, Waters, Miliford,Mass.).

Example 2 Comparison of Current Clinical Grade and New Composition

The CM101 obtained by the method of the present invention is improvedover the current clinical grade CM100. Particularly, the HPLC elutionprofile of FIG. 10 as compared with FIG. 7a illustrates higher purity inthe sample produced according to the present invention. FIG. 7a showsone narrow and symmetric main peak, instead of several peaks.

To further demonstrate the advantageous use of the HIC column and toprovide further evidence of purification of the toxin known as CM101,current clinical grade CM100 was subjected to an HIC column and HPLCpurification and a sugar analysis was performed. The results, in FIG.11, may be compared to FIG. 9. The sugar analysis shows quantitativelyand qualitatively similar end products, and demonstrates that the HICchromatography process removes sugars not related to biologically activeCM100. This result is borne out in Table 3, as well.

TABLE 3 Carbohydrate Composition of CM101 (Presented as IntegralCarbohydrate Ratios) CM101 Purified Current Clinical With PresentCurrent Clinical Grade CM101 Method Grade CM101 Further Purified SugarRhamnose 0 3 0 Mannose 1 3 1 Galactose 3 24 3 Glucose 1 7 1 Glucos- 1 131 amine* Galactos- 1 5 1 amine* *Present as “N-acetyl-glucosamine andN-acetyl-galactosamine”, respectively, in the native polysaccharide.

The table of sugar residues presented above gives approximate molarratios. The actual residues of the CM100 purified according to thepresent method (the first column) are in a range of (0.2-1mannose):(2.5-3.5 galactose):(0.5-1 glucose):(1 N-acetylglucosamine):(0.5-1 N-acetyl galactosamine). The numbers are normalizedto N-acetyl glucosamine: thus N-acetyl glucosamine is set at 1.

For further comparison, FIGS. 12a-b show HPLC profiles of CM100manufactured by the old process (FIG. 12a) and according to the methodof the present invention (FIG. 12b). The gel filtration column (Ultragel100, Waters, Milford, Mass.) was developed in 10 mM phosphate buffer. pH8.4. As seen in FIG. 12b. the CM101 purified according to the method ofthe invention elutes in a relatively narrow peak over an approximately 5minute range. The elution time is approximately 24 minutes from timezero with a 0.3 ml/min flow rate. The material purified by the oldprocess, by contrast. elutes in a broad peak over approximately 12minutes.

Example 3 Analysis of CM101 by SDS-PAGE/Western Blot

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performedusing a 4-20% gradient gel. CM100 samples in a buffer of 2% SDS, 0.5 MTris-HCl, 5% glycerol,0.05% bromophenyl blue. and 5% β-mercaptoethanolpH 0.8 were incubated at 55° for 10 minutes and applied in a 4% stackinggel to the 4 to 20% SDS-PAGE running gel. The gel was run at 200 Voltsfor 90 minutes.

The gel was developed in a western blotting buffer (25 mM Tris. 192 mMglycine and 20% Methanol) and blotted at 100 Volts for 2 hours. The gelwas blocked with 5% fat-free milk in PBS at room temperature for 1 hourand washed twice with a binding buffer (phosphate buffered saline, 2%fetal bovine serum, and 0.5% TWIN-20 detergent (BBT)), then incubated 1hour with 10 μg/ml of 7A3 (monoclonal antibody to CM100).

The gel was incubated 4 times for 10 minutes with BBT and incubated withthe alkaline phosphatase conjugated anti-mouse antibody for 45 minutes,washed 4 times with BBT and developed with Pierce Single-Step APdeveloper for 50 minutes.

The SDS-PAGE/Western Blot analysis suggested that CM100 has a componentof 26,000 Daltons or a multiple thereof when analyzed under theseconditions.

Thus. the method of the present invention provides an improved method ofpurification which minimizes the difficulty of hazardous steps andprovides excellent purity. Additionally, the product produced by themethod taught herein is improved over the currently available CM100.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The invention now being fully described. it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the appendedclaims.

What is claimed is:
 1. A method of purifying a polysaccharide toxin fromgroup B β-hemolytic Streptococcus (GBS) bacteria, which methodcomprises: (a) contacting an aqueous mixture containing the toxin with ahydrophobic interaction chromatography (HIC) resin, (b) separating thetoxin from the HIC resin, (c) containing the toxin with an ion-exchangeresin, and (d) separating the toxin from the ion-exchange resin, whereinthe toxin resulting from the step of separating the toxin from theion-exchange resin is at least approximately 95% pure.
 2. Apolysaccharide toxin from group B β-hemolytic Streptococcus (GBS)bacteria purified by a method which comprises: (a) contacting an aqueousmixture containing the toxin with a hydrophobic interactionchromatography (HIC) resin, and (b) separating the toxin from the HICresin, wherein the toxin is greater than 60% pure after steps (a) and(b).
 3. The toxin of claim 2 wherein the toxin is at least approximately90% pure.
 4. The toxin of claim 3 wherein the is at least approximately95% pure.
 5. The toxin of claim 2 further comprising sugar residues ofmannose, galactose, glucose, N-acetyl glucosamine, and N-acetylgalactosamine in a molar ratio of (0.2-1 mannose):(2.5-3.5galactose):(0.5-1 glucose):1 N-acetyl glucosamine):(0.5-1 N-acetylgalactosamine).
 6. The toxin of claim 5 wherein the sugar residues ofmannose, galactose, glucose, N-acetyl glucosamine, and N-acetylgalactosamine are present in an approximate molar ratio of (1mannose):(3 galactose):(1 glucose):(1 N-acetyl glucosamine):(1 N-acetylgalactosamine).
 7. The toxin of claim 2 having a molecular weight ofapproximately 300,000 Daltons as measured by gel filtrationchromatography in non-denaturing conditions.
 8. The toxin of claim 2characterized in that the toxin elutes in a symmetrical peak measured at203 nm absorbance at approximately 16 minutes from time zero whenanalyzed by HPLC in a 10% acetonitrile buffer with a flow rate ofapproximately 0.3 ml/minute.
 9. The toxin of claim 2 characterized inthat the toxin elutes in a symmetrical peak measured at 203 nmabsorbance at approximately 24 minutes from time zero when analyzed byHPLC in a 10 mM phosphate buffer, pH 8.4.
 10. The toxin of claim 2having a specific activity approximately two to three times greater thanthe specific activity of a GBS toxin that has not had contact with anHIC resin as measured by an assay for increased pulmonary arterialpressure in sheep.
 11. A composition consisting essentially ofpolysaccharide toxin from group B β-hemolytic Streptococcus (GBS)bacteria, wherein the toxin is greater than 60% pure.
 12. Thecomposition of claim 11 wherein the toxin is at least approximately 90%pure.
 13. The composition of claim 12 wherein the toxin is at leastapproximately 95% pure.
 14. The composition of claim 11 wherein thetoxin further comprises sugar residues of mannose, galactose, glucose,N-acetyl glucosamine, and N-acetyl galactosamine in a molar ratio of(0.2-1 mannose):(2.5-3.5 galactose):(0.5 -1 galactosamine in aglucosamine):(0.5-1 N-acetyl galactosamine).
 15. The composition ofclaim 14 wherein the sugar residues of mannose, galactose, glucose,N-acetyl glucosamine, and N-acetyl galactosamine are present in anapproximate molar ratio of (1 mannose):(3 galactose):(1 glucose):(1N-acetyl glucosamine):(1 N-acetyl galactosamine).
 16. The composition ofclaim 11 wherein the toxin has a molecular weight of approximately300,000 Daltons as measured by gel filtration chromatography innon-denaturing conditions.
 17. The composition of claim 11 wherein thetoxin elutes in a symmetrical peak measured at 203 nm absorbance atapproximately 16 minutes from time zero when analyzed by HPLC in a 10%acetonitrile buffer with a flow rate of approximately 0.3 ml/minute. 18.The composition of claim 11 wherein the toxin elutes in a symmetricalpeak measured at 203 nm absorbance at approximately 24 minutes from timezero when analyzed by HPLC in a 10 nM phosophate buffer, pH 8.4.
 19. Apharmaceutical composition comprising polysaccharide toxin from group Bβ-hemolytic Streptococcus (GBS) bacteria and a pharmaceuticallyacceptable carrier, wherein the toxin is greater than 60% pure.
 20. Amethod of treating a patient with a tumor comprising administering thepharmaceutical composition of claim 19 to the patient.
 21. Apolysaccharide toxin from group B β-hemolytic Streptococcus (GBS)bacteria, wherein the toxin is greater than 40% pure and the toxincomprises sugar residues of mannose, galactose, glucose, N-acetylglucosamine, and N-acetyl galactosamine are present in a molar ratio of(0.2-1 mannose):(2.5-3.5 galactose):(0.5-1 glucose):(1 N-acetylglucosamine):(0.5-1 N-acetyl galactosamine).